The outer membrane (OM) of gram-negative bacteria serves as an effective barrier against the permeation of small molecules, thereby protecting the cell from noxious compounds encountered in the environment. In order to obtain the necessary molecules and ions required for growth and function of cell, transport channels are present within the OM. In most gram-negative bacteria such as E. coli, the majority of small molecules are taken up by porins, such as OmpF and OmpC. Porins are non-specific channels and do not bind their substrates. They form water-filled pores within the OM, through which small molecules diffuse, driven by their concentration gradient. The gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen and most prevalently associated with lung infections in cystic fibrosis patients. A severe problem in the treatment of patients infected with P. aeruginosa is the high intrinsic antibiotics resistance of this bacterium. An important reason for the high antibiotics resistance is the low permeability of the OM, due to the absence of porins. In the absence of porins, substrate specific channels belonging to the OprD family are responsible for uptake of the majority of low molecular weight compounds in P. aeruginosa. OprD family members are closely related in sequence, yet transform very different substrates. The archetype of the family, OprD, functions as an uptake channel for basic amino acids and carbapenem antibiotics. No structural information is available for any OprD family member, and the basis for substrate selectivity and specificity within the OprD family is unknown. In this proposal we will determine the X-ray crystal structures of a number of OprD-family channels and characterize their substrate specificity using a range of computational, biochemical and biophysical experiments. More specifically, we will pursue the following aims: 1. Mechanism of substrate binding and transport of P. aeruginosa OprD. 2. Structure determination of a number of OprD family members at high resolution using X-ray crystallography. 3. Biochemical and biophysical characterization of OprD members with determined structures (aim 2). The OprD family provides an excellent opportunity to investigate the structural and biochemical basis for substrate specificity of closely related OM channel proteins. Such an analysis will be the first of its kind for membrane proteins and will provide, besides fundamental knowledge, information that can be used to design more effective drugs against P. aeruginosa. Project narrative Bacterial cells are surrounded by membranes that protect them from harmful and toxic compounds from the outside world. In order for the bacteria to grow, however, small nutrient molecules have to be transported across the membrane into the cell. For this task the bacteria are equipped with specialized proteins in the cell membrane that function as channels and transporters, and which ferry small molecules from the outside of the cell to the inside. For different types of molecules that have to be transported, the bacteria have different families of channel proteins. One such family is the OprD family, members of which occur in a number of human-disease causing bacteria. How members of the OprD family perform their transport tasks is an unanswered question. This proposal aims to answer that question by determining the structures of these proteins at an atomic level by X-ray crystallography. We will also determine what kind of molecules can be transported by members of the OprD family by computational, biochemical and biophysical experiments. Together, this information should enable us to understand how OprD family members perform their tasks in the bacterial membrane. Besides being important from a fundamental point of view, such knowledge could also be used by pharmaceutical companies to design better drugs against disease-causing bacteria.